The advent of combinatorial chemistry techniques has enabled the identification of extremely high numbers of compounds that have potential as therapeutic agents. However, assays for drug metabolism that can rapidly identify those candidate compounds which have a lower potential for rapid metabolic degradation (i.e. short biological half-life) or drug-drug interaction have lagged behind the pace of synthesis and screening of pharmalogical activities. Thus, there is a long-felt need for high throughput assays to assess susceptability of candidate compounds to metabolic degradation, particularly oxidative metabolism, that can rapidly identify suitable candidate compounds (i.e. metabolically stable) for further testing as therapeutic agents.
Of particular interest is the cytochrome P450 (CYP) superfamily of enzymes. The CYP enzymes catalyze reactions which have profound effects on the biological activities of drugs, environmental chemicals and endogenous compounds (Guengerich, FASEB J 6:667-668 (1992); Eastabrook, FASEB J 10:202-204 (1996); and Rendic and Di Carlo, Drug Metab. Rev. 29:413-580 (1997)). In recent years, advances in the study of CYP's by enzyme purification and characterization, gene cloning, and heterologous expression have indicated that five CYP isoforms, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, appear to be most commonly responsible for the metabolism of drugs in humans (Spatnegger and Jaeger, Drug Metab. Rev. 27:397-417 (1995)). Moreover, a number of CYP superfamily enzymes have been successfully expressed in bacterial, yeast, insect and mammalian cells, and have been used to identify substrates and/or inhibitors of these major CYP enzymes (Guengerich and Shimada, Chem. Res. Toxicol. 4:391-407 (1991); Birkett et al., Trends Pharmacol. Sci. 14:151-185 (1993); and Wrighton et al., Drug Metab. Rev. 25:453-484 (1993)).
To date, CYP metabolic activity, and/or inhibition thereof, has been assessed in most cases by performing in vitro incubation using cDNA-expressed enzymes or human liver microsomes (Parkinson, Toxicol. Pathol. 24:45-57 (1996)). Such assessments have required the development and use of assays for quantitative analysis of the parent drug molecules, or the metabolites thereof, which is time-consuming, labor-intensive and costly.
A journal article by Crespi et al. entitled "Microtiter Plate Assays for Inhibition for Human, Drug-metabolizing Cytochrome P450", Anal. Biochem. 248:188-190 (1997) describes a CYP inhibitor assay which utilizes microtiter plate-based fluorometric methods for several major xenobiotic-metabolising CYP isoenzymes, such as CYP3A4 and CYP1A2. Similar assays are also described in Kennedy and Jones, Anal. Biochem. 222:217-223 (1994) and in Donato et al., Anal. Biochem. 213:29-33 (1993). These assays utilize a known substrate for each of the major CYP isoenzymes as a model substrate. The ability of potential drug candidates to interact with CYP enzymes is ranked based on the relative inhibitory effect on metabolism of the model substrates. These assays are relatively fast as compared to other known methods, such as HPLC. However, these assays suffer from significant limitations in that they cannot directly assess the metabolic stability of candidate compounds.
In summary, prior art assays for susceptibility to oxidative metabolism, particularly CYP-mediated metabolism, rely on the measurement of the substrate and/or metabolite(s) in a reaction mixture. Such assays measure the presence of substrate and/or metabolite as a function of time and are thus limited to screening of one compound (or a best a few compounds) at a time. Therefore, what is needed is a rapid, high throughput assay which enables the screening of many compounds for susceptibility to oxidative metabolism in a single effort.